Suicide is a major public health concern as about 35,000 people die of suicide in the U.S. every year. Recent studies of patients with suicidal behavior and of postmortem brain samples of suicide victims strongly suggest that suicide is associated with neurobiological abnormalities, such as abnormalities in serotonin function. It has also been observed that suicide and suicidal behavior are associated with abnormal hypothalamic-pituitary- adrenal (HPA) axis function. The molecular mechanisms and causes of HPA axis abnormalities in suicide, however, are not well understood. It is believed that HPA axis abnormalities may be related to an abnormal feedback mechanism and excessive secretion of corticotropin releasing factor (CRF) in the brain. The main objective of our proposal is to examine if HPA axis genes are abnormally expressed in the prefrontal cortex (PFC), anterior cingulate cortex/gyrus (CG), hippocampus, and amygdala of suicide victims. To achieve this specific aim, we will determine the protein and mRNA expression of the HPA axis genes, glucocorticoid receptors (GR-? and GR-?), mineralocorticoid receptors (MR), CRF, the receptors for CRF (CRF-R1 and CRF- R2), CRF binding protein (CRF-BP), and the target genes for GR, which are GILZ and FKBP in the PFC, CG, hippocampus, and amygdala of adult suicide victims of different diagnoses (depression, schizophrenia, other suicide) and in non-suicidal patients with different diagnoses (depression, schizophrenia) as well as normal controls. Abnormalities of epigenetic regulation have been implicated in the regulation of some of the HPA axis genes, especially the GR. We therefore propose to study epigenetic regulation of these genes by examining the effects of DNA methylation on the regulation of HPA axis genes. We propose to determine the protein and mRNA expression of enzymes that methylate DNA (DNA methyl transferase 1 [DNMT1], DNMT3a, and DNMT3b) and hydroxmenthylate DNA (TET1, TET2, and TET3) in the PFC, CG and hippocampus of adult suicide victims, non-suicidal subjects and normal control subjects. We also propose to determine the DNA methylation and hydroxymethylation of promoter proximal regions of HPA axis genes in the PFC, CG and hippocampus of adult suicide victims, non-suicidal patients and controls. We will use methyl DNA immunoprecipitation of DNA (MeDIP) and hydroxymethyl DIP (hMeDIP) to identify differentially ethylated/hydroxymethylated regions of interest in HPA axis genes of adult suicide cohorts. We will validate differences in identified regions using pyrosequencing of DNA isolated from each postmortem region. Together these studies will provide important information to support our hypothesis that suicide is associated with abnormal expression of some of the HPA axis related genes and with abnormal methylation/hydroxymethylation of HPA axis genes critical to the downstream effects of this stress response. These studies will not only enhance our knowledge of the pathophysiology of suicide in general and the role of HPA axis in suicide in particular, but also may result in identifying important sites that may be used for the development of appropriate therapeutic agents for treatment of suicidal behavior and as biomarkers for suicide.